2,2-bis-(hydroxymethyl)cyclopropylidenemethyl-purines and -pyrimidines as antiviral agents

ABSTRACT

Compounds which are active against viruses have the following formulas: 
                         
wherein B is a purine or pyrimidine heterocyclic ring or base. In a preferred embodiment, the purine include 6-aminopurine (adenine), 6-hydroxypurine (hypoxanthine), 2-amino-6-hydroxypurine (guanine), 2,6-diamino-purine, 2-amino-6-azidopurine, 2-amino-6-halo substituted purines such as 2-amino-6-chloropurine, 2-amino-6-fluoropurine, 2-amino-6-alkoxypurines such as 2-amino-6-methoxypurine, 2-amino-6-cyclopropylaminopurine, 2-amino-6-alkylamino or 2-amino-6-dialkylamino substituted purines, 2-amino-6-thiopurine, 2-amino-6-alkylthio substituted purines, 3-deazapurines, 7-deazapurines and 8-azapurines. The pyrimidine incorporates cytosine, uracil and thymine, 5-halo substituted cytosines and uracils, 5-alkyl substituted cytosines and uracils including derivatives with a saturated or unsaturated alkyl group and 6-azapyrimidines.

RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 12/139,750, filed Jun. 16, 2008, pending, which is a divisionof U.S. patent application Ser. No. 10/942,313, filed Sep. 15, 2004, nowU.S. Pat. No. 7,393,855, which is a continuation of InternationalApplication No. PCT/US03/007916, filed Mar. 14, 2003, which claims thebenefit of U.S. Provisional Application No. 60/364,518, filed Mar. 15,2002, the content of all of which are incorporated herein by reference.

SPONSORSHIP

Work on this invention was supported in part by the National CancerInstitute, Grant No. RO1 CA32779 and National Institute of Allergy andInfectious Diseases; Grant No. PO1-AI46390. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel purine and pyrimidine compoundswhich have antiviral activity and methods of making and using thosecompounds.

BACKGROUND OF THE INVENTION

Viruses are the etiologic cause of many life-threatening human diseases.Of special importance are herpes viruses such as herpes simplex 1(HSV-1), herpes simplex 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barrvirus (EBV), varicella zoster virus (VZV) and human herpes viruses 6, 7and 8 (HHV-6, -7 and -8) which are associated with many common viralillnesses. The HSV-1 and HSV-2 infections are characterized by coldsores of skin, mouth or genital region. After primary infection, thevirus is harbored in neural cells and can reappear later in the life ofa patient. Human CMV (HCMV) infection is a life-long affliction whichcan result in morbidity and mortality. These pathologies includemicrocephaly, hepatosplenomegaly, jaundice, encephalitis, infections ofthe newborn infants or fetuses in utero, and infections ofimmunocompromised hosts. HCMV infection is responsible for retinitis,gastritis and pneumonitis in AIDS patients and HCMV-induced pneumoniasor hepatitis are frequent and serious complications of organ or bonemarrow transplants. EBV causes infectious mononucleosis and it isconsidered as the etiologic agent of nasopharyngeal cancer,immunoblastic lymphoma, Burkitt's lymphoma and hairy leukoplakia. VZVcauses chicken pox and shingles. Although in children the chicken pox isusually a non-fatal disease, the recurrent form of this infection,shingles, may in advanced stage lead to paralysis, convulsions andultimately death. Again, in immunocompromised patients the infectionwith VZV is a serious complication. Human herpes virus 6 (HHV-6) whichis commonly associated with children's rash was also identified inacquired immunodeficiency syndrome (AIDS) patients and it may be acofactor in the pathogenesis of AIDS in hosts infected with humanimmunodeficiency virus (HIV). Levine, A. J. Viruses, Ch. 4, W. H.Freeman & Co., New York, pp. 67-85 (1992); Human Herpesvirus Infections,Raven Press, New York (1986); Schirmer, E. C., et al., Proc. Natl. Acad.Sci. USA 88:5922-5926 (1992). Human herpes virus 8 (HHV-8) wasidentified in patients with Kaposi sarcoma, a fatal afflictionaccompanying AIDS. Chang, Y., et al., Science 266:1865-1869 (1994).

HIV is the underlying cause of AIDS, a world-wide epidemic with fatalconsequences. According to the Joint United Nations Programme onHIV/AIDS, 40 million people are estimated to be living with HIV/AIDS atthe end of 2001. During that same year, AIDS caused the deaths of anestimated 3 million people.

Hepatitis B virus (HBV) is a virus that causes chronic diseaseresponsible for serious liver damage, including cirrhosis of the liver,cancer, organ failure and ultimately, death. It is estimated thatapproximately 300 million people worldwide are infected with HBV.According to the CDC, there are approximately 1.25 million Americanschronically infected with HBV. Although use of a prophylactic vaccinehas reduced the incidence of new HBV infections, there continues to be aneed for an effective therapeutic drug.

Various derivatives of nucleoside analogues have been found to exhibitantiviral activity. Notably, acyclovir (Zovirax) and its prodrugvalacyclovir (Valtrex) are approved drugs for infections caused by HSV-1and HSV-2. Acyclovir Therapy for Herpesvirus Infections (Baker, Ed.), M.Dekker, New York (1990); Against HCMV, four drugs are currentlyavailable: Ganciclovir (Cytovene), cidofovir (Vistide), antisenseoligonucleotide fomivirsen (Vitravene) and foscarnet (Foscavir).However, only ganciclovir is effective orally but it requires largedoses and produces potentially serious adverse effects such as bonemarrow suppression. Ganciclovir Therapy for Cytomegalovirus Infection(Spector, S. S., Ed.), M. Dekker, New York (1991). A considerable effortwent into design, synthesis and biological investigation of analogues ofthese drugs as well as in development of new antiviral agents. Larsson,A., et al., Antimicrob. Agents & Chemother. 30:598-605 (1986); Ashton,W. T., et al., J. Med. Chem. 31:2304-2315 (1988). Cidofovir andfomivirsen are approved only for topical application against retinitisin AIDS patients and foscarnet is used only by intravenous route and itleads to characteristic toxicity.

Current drugs for AIDS include AZT (zidovudine, Retrovir), ddI(didanosine, Videx), ddC (zalcitabine, Hivid) and d4T (stavudine,Zerit). De Clercq, E., J. Med. Chem. 38:2491-2517 (1995). Allenicnucleoside analogues such as adenallene and cytallene are examples ofanti-HIV agents containing an unsaturated alkyl group. U.S. Pat. No.4,935,427; Zemlicka, J., Allenols Derived from Nucleic Acid Bases—a NewClass of Anti-HIV Agents: Chemistry and Biological Activity inNucleosides and Nucleotides as Antitumor and Antiviral Agents (Chu, C.K.; Baker, D. C., Eds.), Plenum Press, New York, pp. 73-100 (1993). ForHBV, alpha interferon and 3TC (lamivudine; Epivir) are two drugslicensed for the treatment of persons with chronic HBV infection.Unfortunately, only about 40% of patients respond to these drugs andresistance is a growing problem.

Particular 2-hydroxymethylcyclopropylidenemethylpurines and theirutility against certain viruses have been described elsewhere (see, forexample, co-owned U.S. Pat. No. 6,352,991; Qiu, Y. L., et al., J. Med.Chem. 41:10-23 (1998); Antiviral Chem. Chemother. 9:341-352 (1998)).However, there continues to be a need for novel compounds which areactive against pathogenic viruses, including HCMV, HSV-1, HSV-2, HHV-6,HIV, and hepatitis B virus (HBV).

SUMMARY OF THE INVENTION

The present invention describes novel2,2-bis-(hydroxymethyl)cyclopropylidenemethyl derivatives andheterocyclic compounds, prodrugs and pharmacologically acceptable saltsthereof. These compounds which have been found to be useful antiviralagents and are effective against HCMV, HSV-1, HSV-2, HIV, EBV and HBV,as well as against other mammalian viruses. The compounds of the presentinvention have the following Formulas:

wherein B is a purine or pyrimidine heterocyclic ring or base. In apreferred embodiment, the purine includes 6-aminopurine (adenine),6-hydroxypurine (hypoxanthine), 2-amino-6-hydroxypurine (guanine),2,6-diamino-purine, 2-amino-6-azidopurine, 2-amino-6-halo substitutedpurines such as 2-amino-6-chloropurine, 2-amino-6-fluoropurine,2-amino-6-alkoxypurines such as 2-amino-6-methoxypurine,2,6-diaminopurine, 2-amino-6-cyclopropylaminopurine,2-amino-6-alkylamino or 2-amino-6-dialkylamino substituted purines,2-amino-6-thiopurine, 2-amino-6-alkylthio substituted purines,3-deazapurines, 7-deazapurines and 8-azapurines. The pyrimidineincorporate cytosine, uracil and thymine, 5-halo substituted cytosinesand uracils, 5-alkyl substituted cytosines and uracils includingderivatives with a saturated or unsaturated alkyl group and6-azapyrimidines.

Compositions useful for treatment of viral infections, such as HCMV,HSV-1, HSV-2, HHV-6, HIV, EBV and HBV contain an effective amount of atleast one compound of Formulas 1 to 2 or a pharmaceutically acceptablesalt thereof.

The present invention also includes a method for synthesizing compoundsof Formulas 1 and 2 wherein B is a heterocyclic ring derived from purineor pyrimidine moiety such as 6-aminopurine (adenine), 6-hydroxypurine(hypoxanthine), 2-amino-6-hydroxypurine (guanine), 2,6-diaminopurine,2-amino-6-azidopurine, 2-amino-6-halo substituted purines such as 2-amino-6-chloropurine, 2-amino-6-fluoropurine, 2-amino-6-alkoxypurinessuch as 2-amino-6-methoxypurine, 2,6-diaminopurine,2-amino-6-cyclopropyl-aminopurine, 2-amino-6-alkylamino or2-amino-6-dialkylamino substituted purines, 2-amino-6-thiopurine,2-amino-6-alkylthio substituted purines, 3- and 7-deazapurines such as3- and 7-deazaadenine, 8-azapurines such as 8-azaadenine; cytosine,uracil, 5-halocytosine and 5-halouracil and related alkyl derivativescontaining a saturated or unsaturated alkyl group at the 5-position),thymine, 6-azapyrimidines such as 6-azacytosine and wherein the alkylside-chain attached to the heterocyclic ring is a2,2-bis-(hydroxyl-methyl)cyclopropylidene methane moiety.

Additional objects, advantages, and features of the present inventionwill become apparent from the following description, taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent toone skilled in the art by reading the following specification and byreferencing the following drawings in which:

FIGS. 1 and 2 show two procedures for the synthesis of1,1-dibromo-methyl-2,2-bis-(acetoxymethyl)cyclopropane (3);

FIG. 3 shows the synthesis of (Z)- and(E)-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purines and-pyrimidines of Formulas 1 and 2;

FIG. 4 shows the method for separation of(Z)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}cytosine (1e) and(E)-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}cytosine (2e);

FIG. 5 shows the hydrolysis of(Z)-2-amino-6-chloro-9-{[2,2-bis-(hydroxylmethyl)cyclopropylidene]methyl}purine(1b) to (Z)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}guanine(1c);

FIG. 6 shows the hydrolysis of(E)-2-amino-6-chloro-9-{[2,2-bis-(hydroxylmethyl)cyclopropylidene]methyl}purine(2b) to (E)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}guanine(2c);

FIG. 7 shows the synthesis of(Z)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}thymine (1f) and(E)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}thymine (2f);

FIG. 8 shows the synthesis of(Z)-2-amino-6-methoxy-9-{[2,2-bis-(hydroxylmethyl)cyclopropylidene]methylpurine(1g) and(E)-2-amino-6-methoxy-9-{[2,2-bis-(hydroxylmethyl)cyclopropylidene]methylpurine(2g);

FIG. 9 shows the synthesis of(Z)-2-amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxyl-methyl)cyclopropylidene]methylpurine(1h) and(E)-2-amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(2h);

FIG. 10 shows the synthesis of(Z)-2,6-diamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(1o) and(E)-2,6-diamino-9-([2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(2o); and

FIG. 11 shows the synthesis of(Z)-2-amino-6-fluoro-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(1p) and(E)-2-amino-6-fluoro-9-{[2,2-bis-(hydroxylmethyl)cyclopropylidene]methylpurine(2p).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the following terms shall be defined as follows (unlessotherwise noted):

“Alkyl” shall mean a saturated straight chain or branched, primary,secondary, or tertiary hydrocarbon radical that is fully saturated,typically C₁-C₁₈, preferably C₁-C₁₀, and more preferably C₁-C₆ Preferredalkyl groups include, without limitation, methyl, ethyl, propyl, butyl,pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl, isopentyl, amyl,and t-pentyl. For the purposes of this invention, any carbon in thealkyl moiety may be replaced with oxygen (O), sulfur (S), or nitrogen(N).

“Cycloalkyl” shall mean a mono-, bi- or polycyclic alkyl radical. Forconvenience, the term “cycloalkyl” shall also expressly includecycloalkenyl cycloalkynyl radicals. A “branched cycloalkyl” shall mean acycloalkyl ring in which one or more ring members are substituted withalkyl. In general, these rings shall typically be C₃-C₁₈, preferablyC₃-C₁₀, and more preferably C₃-C₈.

“Halo” shall mean fluoro, chloro, bromo, or iodo.

“Heterocyclyl” shall mean a mono-, bi- or polycyclic radical containingone or more rings which may be saturated, unsaturated, or aromatic,wherein at least one ring contains one or more heteroatoms selected fromnitrogen (N), oxygen (O), and sulfur (S). Heterocyclyl radicalstypically have 3-18 total ring members and preferably 3-10 total ringmembers. Preferably, heterocyclyl radicals are monocyclic (preferablyhaving 3-8 and more preferably, 3-6 ring members) or bicyclic(preferably having 6-12 ring members and more preferably, 8-10 ringmembers). Suitable heterocyclyl for use in the compounds of thisinvention include radicals of (without limitation) furan, dioxolane,thiophene, pyrrole, pyrazole, triazole, imidazole, pyrrolidine, pyran,pyridine, pyrimidine, morpholine, piperidine, oxazole, isoxazole,oxazoline, oxazolidine, oxathiazole, thiazole, isothiazole, thiadiazole,tetrazole, benzofuran, indole, isoindole, quinazoline, quinoline,isoquinoline, purine, pyrrolopyrimidine, pyrrazolopyrimidine, pteridine,ketal. In addition, heterocyclyl radicals may contain one or moresubstituents (i.e., a ring substituent, such as a halogen atom, an alkylradical, or aryl radical) attached to a ring member atom of theheterocyclyl radical. All stable isomers of heterocyclyl groups arecontemplated in this definition.

“Lower” shall mean the group to which it is applied preferably has 1-6,and more preferably 1-4, carbon atoms, except in the case of rings (suchas cycloalkyl and heterocyclyl), in which case “lower” signifies 3-6ring members.

“Patient” shall mean any warm-blooded mammal, including withoutlimitation, a human.

“Pharmaceutically acceptable salts” shall mean those salts of anycompound of this invention derived from an inorganic or organic acid orbase recognized in the art as compatible for pharmaceuticalcompositions. Examples of suitable acids include hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic and benzenesulfonic acids. Other acids such asoxalic, while not in themselves pharmaceutically acceptable, may beuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium, potassium),alkaline earth metal (e.g., magnesium), ammonium and NR₄ ⁺ (where R is aC₁₋₄ alkyl) salts, and the like. Reference to a compound according tothe invention is understood to include any and all correspondingpharmaceutically acceptable salts thereof. For convenience, the terms“pharmaceutical” and “pharmaceutically acceptable” are understood toencompass compounds acceptable for the practice of veterinary medicineas well.

“Pharmaceutically acceptable carriers” for use in the formulations ofthis invention are carriers that are compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

“Therapy” and “therapeutic” shall mean treatment of an individual for aviral infection or disease. For convenience, these terms are alsounderstood to encompass prophylactic or precautionary use oradministration of a compound of this invention. Such precautionary orprophylactic use is exemplified by administration of an antiviral agentto an individual(s) suspected, but not proven, of having a viralinfection or to an individual(s) susceptible to contracting a pathogenicviral infection due to contact with contaminated items, or contact withother individuals carrying a contagious viral disease.

All published documents referred to herein are expressly incorporatedherein by reference.

The compounds of the present invention which have been found to beeffective against herpes viruses such as human cytomegalovirus (HCMV)and herpes simplex virus type 1 (HSV-1), human immunodeficiency virusand hepatitis B virus, are compounds of Formulas 1 to 2, wherein Brepresents a heterocyclic ring derived from a purine moiety (preferablyattached via the 9-position) or pyrimidine moiety (preferably attachedvia the 1-position) such as 6-aminopurine (adenine), 6-hydroxypurine(hypoxanthine), 2-amino-6-hydroxypurine (guanine), 2,6-diamino-purine,2-amino-6-azidopurine, 2-amino-6-halo substituted purines such as2-amino-6-chloropurine, 2-amino-6-fluoropurine, 2-amino-6-alkoxypurinessuch as 2-amino-6-methoxypurine, 2,6-diaminopurine,2-amino-6-cyclopropylaminopurine, 2-amino-6-alkylamino or2-amino-6-dialkylamino substituted purines, 2-amino-6-thiopurine,2-amino-6-alkylthio substituted purines, 3- and 7-deazapurines such as3- and 7-deazaadenine, 8-azapurines such as 8-azaadenine; cytosine,5-halocytosine and 5-halouracil (wherein halo is bromo, chloro, iodo orfluoro) and related alkyl derivatives containing a saturated orunsaturated alkyl group at the 5-position), thymine, 6-azapyrimidinessuch as 6-azacytosine, wherein the alkyl side-chain attached to theheterocyclic ring is a 2,2-bis-(hydroxymethyl)cyclopropylidene-methanemoiety.

The preferred compounds of the present invention are(Z)-9-{[2,2-bis-(hydroxyl-methyl)cyclopropylidene]methyl}adenine (1a),(Z)-9-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}guanine (1c),(Z)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}cytosine (1e),(Z)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}thymine (1f),(E)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}adenine (2a),(E)-9-{[2,2-bis-(hydroxylmethyl)cyclopropylidene]methyl}guanine (2c),(Z)-2-amino-6-methoxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(1g) and(E)-2-amino-6-methoxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(2g). The preferred compounds of the present invention are also(Z)-2-amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(1h),(E)-2-amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(2h),(Z)-2-amino-6-allylamino-9-[{2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(11),(E)-2-amino-6-allylamino-9-([2,2-bis-(hydroxymethyl)cyclopropylidene]methyl)-purine(21),(Z)-2-amino-6-propargylamino-9-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}purine(1j),(E)-2-amino-6-propargylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(2j),(Z)-2-amino-6-cyclopropylmethylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(1k),(E)-2-amino-6-cyclopropylmethylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(2k),(Z)-2-amino-6-propyloxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(1 I),(E)-2-amino-6-propyloxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(21),(Z)-2-amino-6-allyloxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(1 m),(E)-2-amino-6-allyloxy-9-{[2,2-bis-(hydroxymethyl)-cyclopropylidene]methyl}purine(2m),(Z)-2-amino-6-propylthio-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine(1n) and(E)-2-amino-6-propylthio-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(2n);(Z)-2,6-diamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(1o),(E)-2,6-diamino-9-[2,2-bis-(hydroxymethyl)-cyclopropylidene]methylpurine(2o),(Z)-2-amino-6-fluoro-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(1p); and(E)-2-amino-6-fluoro-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(2p).

The nomenclature of the compounds of the present invention followsstandard conventions. The numbering of the cyclopropylidenemethanemoiety attached to the heterocyclic ring B is shown in Formulas 1 and 2.The purine and pyrimidine rings are numbered as shown below:

It is appreciated that heterocyclic rings containing hydroxy and aminogroups are tautomeric with the corresponding oxo and imino compounds.For the sake of clarity, it is noted that Formula 1 is the Z-isomer andFormula 2 is the E-isomer of the novel2,2-bis-(hydroxyl-methyl)cyclopropylidenemethyl compounds of thisinvention.

The syntheses of exemplified compounds of the present invention aresummarized in FIGS. 1 to 8. Generally, suitably O-protected1-halo-1-halomethyl-2,2-bis-(hydroxymethyl)cyclopropanes can serve asalkylating agents. The preferred reagent,1,1-dibromomethyl-2,2-bis-(acetoxymethyl)-cyclopropane (3), was preparedas shown in FIG. 1. Commercially available diethylisopropylidene-malonate (4) was converted to bromo derivative 5.Compound 5 was then transformed to a 1:1 mixture of diethylisopropylidene malonate (4) and diethyl methylene-cyclopropane1,1-dicarboxylate (6) by a modification of the described procedure.Ullman, E. F., J. Am. Chem. Soc. 81:5386-5392 (1959). Compounds 4 and 6were separated by chromatography on a silica gel column and diethylmethylene-cyclopropane 1,1-di-carboxylate (6) was reduced to2,2-bis-(hydroxymethyl)methylenecyclopropane (7) by lithium aluminumhydride in ether as described by Dolbier, W. R., et al., J. Am. Chem.Soc. 93:3933-3940 (1971). Acetylation with acetic anhydride in pyridineprovided 1,1-bis-(acetoxymethyl)methylenecyclopropane (8). Addition ofelemental bromine in a suitable solvent such as carbon tetrachloridegave 1,1-dibromomethyl-2,2-bis-(acetoxymethyl)-cyclopropane (3).

Alternately, as shown in FIG. 2, a mixture of diethyl isopropylidenemalonate (4) and methylenecyclopropane-2,2-dicarboxylate (6) was reducedwith lithium aluminum hydride to give 2-isopropylidenepropane-1,3-diol(9) and 2,2-bis-(hydroxymethylene)methylene-cyclopropane (7).Acetylation provided the corresponding acetates 8+10. Mixtures ofcompounds 7+9 and 8+10 are not separable by chromatography on a silicagel column. In the next step, addition of bromine on compounds 8+10 wasperformed using pyridinium perbromide in dichloromethane to furnish1,1-bis(acetoxymethyl)-1,2-dibromo-2,2-dimethyl-ethane (11) and1,1-dibromomethyl-2,2-bis-(acetoxymethyl)cyclopropane (3) which wereseparated by chromatography on a silica gel column.

The 1,1-dibromomethyl-2,2-bis-(acetoxymethyl)cyclopropane (3) was usedas an alkylating agent in conjunction with an appropriate nucleic acidbase adenine (12a) or precursor 2-amino-6-chloropurine (12b) orN⁴-acetylcytosine (12d). Alkylation effected by potassium carbonate inan organic solvent, e.g., N,N-dimethylformamide, at an elevatedtemperature, e.g., 100° C., was accompanied by elimination of elementsof hydrogen bromide to give the isomeric mixtures of (Z)- and(E)-9-{[2,2-bis-(acetoxymethyl)cyclopropylidene]-methyl}adenines 13a and14a or (Z)- and(E)-2-amino-6-chloro-9-{[2,2-bis-(acetoxymethyl)cyclopropylidene]-methyl}purines13b and 14b or (Z)- and(E)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}cytosines 1e and2e. In the latter case, N-acetyl and O-acetyl groups were removed by awork-up of the reaction mixture with methanol at an elevatedtemperature. Deacetylation of intermediates 13a+14a or 13b+14b wasperformed with potassium carbonate in aqueous methanol to give the (Z)-and (E)-9-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}-adenines 1aand 2a or (Z)- and(E)-2-amino-6-chloro-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purines1b and 2b which were separated by chromatography on silica gel (FIG. 3).

Because mixture of 1e+2e was not separable by chromatography, it wasconverted to N⁴-benzoyl derivatives 15 and 16 by benzoic anhydride inethanol. This method was used previously for the1-[(2-hydroxymethyl)cyclopropylidenemethyl]cytosines. Qiu, Y.-L., etal., Antiviral Chem. Chemother. 9:341-352 (1998). The (Z)- and(E)-N⁴-benzoyl derivatives 15 and 16 were separated by chromatography onsilica gel. The individual isomers 15 and 16 were debenzoylated withammonia in methanol to afford(Z)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}cytosine (1e) and(E)-1-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}cytosine (2e)(FIG. 4).

Hydrolysis of separated compounds 1b and 2b with formic acid gave(Z)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}guanine (1c)(FIG. 5) and(E)-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}guanine (2c)(FIG. 6).

For thymine analogues 1f and 2f a different approach was adopted. The2,4-bis-(trimethylsilyloxy)-5-methylpyrimidine (17) (prepared asdescribed by Iwai I., et al., In: Synthetic Procedures in Nucleic AcidChemistry, Vol. 1 (Editors Zorbach, W. W. and Tipson, R. S.), John Wileyand Sons, New York, 1968, pp. 338-394) was refluxed with alkylatingagent 3 in acetonitrile for a prolonged period of time to giveintermediate 18. In a subsequent step, elimination of elements of HBrwas performed using potassium carbonate in N,N-dimethylformamide.Finally, deacetylation with potassium carbonate in aqueous methanolafforded (Z)-1-{[2,2-bis-(hydroxymethyl)-cyclopropylidene]methyl}thymine(1f) and (E)-1-{[2,2-bis-(hydroxymethyl)-cyclopropylidene]methyl}thymine(20 which were separated by chromatography on silica gel (FIG. 7).

Reaction of compound 1b or 2b with methanol and potassium carbonate gave(Z)-2-amino-6-methoxy-9-([2,2-bis-(hydroxymethyl)cyclopropylidene]-methylpurine(1g) or(E)-2-amino-6-methoxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(2g).

Reaction of compound 1b or 2b with cyclopropylamine gave(Z)-2-amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(1h) or(E)-2-amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(2h).

Reaction of compound 1a or 1b with ammonia gave(Z)-2,6-diamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(1o) or(E)-2,6-diamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methylpurine(2o).

Reaction of compound 1b or 2b with less than stoichiometric amount oftrimethylamine and potassium fluoride gave(Z)-2-amino-6-fluoro-9-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methylpurine(1p) or(E)-2-amino-6-fluoro-9-([2,2-bis-(hydroxymethyl)cyclo-propylidene]methylpurine(2p).

Compositions within the scope of invention include those comprising anovel compound of the present invention in an effective amount toachieve an intended purpose. Determination of an effective amount andintended purpose is within the skill of the art. Preferred dosages,which are dependent for example, on the severity of the infection andthe individual patient's response to the treatment, can range from about0.01 to about 100 mg/kg of body weight to give a blood concentrationranging from 0.05 μg to about 5 mg per mL of whole blood.

As used herein, the term “pharmaceutically acceptable salts” is intendedto mean salts of the compounds of the present invention withpharmaceutically acceptable acids, e.g., inorganic acids such assulfuric, hydrochloric, phosphoric, etc. or organic acids such as aceticor succinic.

Pharmaceutically acceptable compositions of the present invention mayalso include suitable carriers comprising excipients and auxiliarieswhich facilitate processing of the active compounds into preparationswhich may be used pharmaceutically. Such preparations, preferably thosewhich can be administered orally, include tablets, dragees and capsules.Further preferred preparations are those which can be administeredrectally, such as suppositories, as well as suitable solutions foradministration by injection or orally, contain about 0.1 to about 99%,preferably 25 to 85%, of the active compound of the present invention,together with the excipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which itself is known, e.g., using theconventional mixing, granulating, dragee-making, dissolving orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, e.g.,lactose or sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, e.g., calcium phosphate, (e.g., tricalciumdiphosphate or calcium hydrogen phosphate) as well as binders such asstarch paste, using, e.g., maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose and/orpolyvinylpyrrolidone. If desired, disintegrating agents may be addedsuch as the above-mentioned starches, carboxymethyl starch,carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, agar, oralginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, e.g., silica,talc, stearic acid or salts thereof, such as magnesium stearate orcalcium stearate, and/or polyethylene glycol. Dragee cores are providedwith suitable coatings which, if desired, are resistant to gastricjuices. For this purpose, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinylpyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvent or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate, are used. Dyestuffs or pigmentsmay be added to the tablets or dragee coatings, e.g., for identificationor in order to characterize different combinations of active compounddoses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules may contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe used.

Possible pharmaceutical preparations which can be used rectally include,e.g., suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, e.g.,natural or synthetic triglycerides, paraffin hydrocarbons, polyethyleneglycols or higher alkanols. It is also possible to use gelatin rectalcapsules which consist of a combination of the active compounds with abase. Possible base materials include, e.g., liquid triglycerides,polyethylene glycols or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, e.g.,water-soluble salts. In addition, suspension of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, such as sesame oil,or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increaseviscosity of the suspension, such as carboxymethylmethylcellulose,sorbitol and/or dextran. Optionally, the suspension may also containstabilizers.

Alternately, the active compounds of the present invention may beadministered in the form of liposomes, pharmaceutical compositionswherein the active compound is contained either dispersed or variouslypresent in corpuscules consisting of aqueous concentrate layers adherentto hydrophilic lipid layer. The active compound may be present both inthe aqueous layer and in the lipidic layer or in non-homogeneous systemgenerally known as lipophilic suspension.

It will be appreciated that the active compounds of the presentinvention may be administered in combination with known antiviralagents, e.g., acyclovir, ganciclovir, foscarnet, cidofovir, fomivirsen,zidovudine, AZT, ddI, ddC, 3TC and d4T.

The present invention also contemplates prodrugs of the compounds ofFormulas 1 and 2. Prodrugs of the antiviral compounds of the presentinvention may include, e.g., lipophilic phosphate esters or amidatescapable of penetrating the cell membrane. Those skilled in the art willappreciate that the aim of prodrugs is to provide effective therapeuticagents for resistant strains of viruses (McGuigan, C., et al., J. Med.Chem. 36:1048-1052 (1993)) or activate inactive analogues. Franchetti,P., et al., J. Med. Chem. 37:3534-3541 (1994). See also “The OrganicChemistry of Drug Design and Drug Action,” Chapter 8, R. B. Silverman,Academic Press (San Diego), 1992.

The following Examples further describe the compounds of the presentinvention and the synthesis schemes for producing same.

EXAMPLE 1 Synthesis of Diethyl Bromoisopropylidenemalonate (5)

Diethyl isopropylidenemalonate (4, 50g, 0.25 mol) was refluxed withstirring with N-bromosuccinimide (44.3 g, 0.25 mol) and dibenzoylperoxide (1.0 g, 4.1 mmol) in carbon tetrachloride (100 mL) withillumination using Kodak Ectagraphic slide projector lamp ELH (300 Watt)for 1.5 h. The reaction was completed as indicated by a negativestarch-iodine test for N-bromosuccinimide. The resulting mixture wasdiluted with carbon tetrachloride (100 mL) and it was cooled in anice-bath. The precipitated succinimide was filtered off and the filtratewas evaporated in vacuo at room temperature. The residual pale yellowoil of diethyl bromo-isopropylidene-malonate (5, 71.1 g) was usedwithout purification in the Example 2.

EXAMPLE 2 Diethyl Methylenecyclopropane-2,2-dicarboxylate (6)

Compound 5 (43.4 g, 0.156 mol) from Example 1 was added to a vigorouslystirred refluxing solution of potassium tert-butoxide (17.5 g, 0.156mol) in tert-butyl alcohol (500 mL) under nitrogen. The stirring wascontinued for 15 min. and the mixture was immediately cooled in an icebath. Acetic acid was then added, the solid portion was filtered off andthoroughly washed with ether. The filtrate was concentrated in vacuo,diluted with ether and the organic layer was washed several times withwater. After drying with magnesium sulfate, the solution was evaporatedin vacuo and the residue was distilled, bp. 99-93° C./0.3 torr, yield14.8 g (47%) of a 1:1 mixture of 4+6. This mixture was chromatographedon a silica gel column using first hexanes-ether (40:1) and then (20:1)to give product 6 (7.3 g, 23%) as a colorless liquid.

¹H NMR (CDCl₃, 300 MHz) δ 1.24 (t, 6H, J=7.2 Hz, CH₃), 2.15 (t, 2H,J=2.4 Hz, H₃), 4.17 (q, 4H, J=7.2 Hz, OCH₂), 5.53 (t, 1H, J=2.1 Hz) and5.62 (s, 1H, J=2.6 Hz, C═CH₂). ¹³C NMR (CDCl₃, 75 MHz) ppm 14.21 (CH₃),18.24 (C₃), 23.26 (C₂), 61.02 (CH₂O), 105.20 (═CH₂), 130.46 (C₁), 167.91(CO).

EXAMPLE 3 2,2-(Bis-hydroxymethyl)methylenecyclopropane (7)

A solution of diethyl methylenecyclopropane-1,1-dicarboxylate (6, 6.50g, 32 mmol) from Example 2 in ether (60 mL) was added to a stirredsuspension of lithium aluminum hydride (1.90 g, 51 mmol) in ether (50mL) at such a rate to maintain a gentle reflux. The resultant mixturewas refluxed for 15 h. It was then quenched carefully with water (4 mL)and 2 M sodium hydroxide (8 mL). The ether phase was separated and theremaining white precipitate was extracted with ether. Ether fromcombined organic phases was distilled off using a Vigreux column to give2,2-bis-(hydroxymethyl)methylenecyclopropane (7, 2.84 g, 78% yield) as aresidue (colorless oil). The ¹H-NMR spectrum was identical to thatdescribed by Dolbier, W. R., et al., J. Am. Chem. Soc. 93:3933-3940(1971).

EXAMPLE 4 2,2-Bis-(acetoxymethyl)methylenecyclopropane (8)

To a solution of compound 7 (2.65 g, 23 mmol) from Example 3 in pyridine(6 mL) acetic anhydride (13 mL) was added dropwise with stirring at roomtemperature. The stirring was continued for 16 h. The reaction wasquenched with water and the product was extracted with cold (4° C.)pentane (70 mL) at 4° C. The combined organic phase was washedsuccessively with saturated aqueous copper sulfate, 5% hydrochloricacid, aqueous sodium hydrogen carbonate and brine. It was then driedwith magnesium sulfate, the solvent was evaporated in vacuo and theresidue was chromatographed on a silica gel column (hexanes-ether, 20:1)to give compound 8 as a colorless liquid (4.28 g, 93%).

¹H NMR (CDCl₃, 300 MHz) δ 1.34 (t, 2H, J=2.1 Hz, H₃), 2.07 (s, 6H, CH₃),4.07 (AB, 4H, J_(AB)=11.6 Hz, OCH₂), 5.46 (t, J=1.8 Hz, 1H) and 5.40 (t,1H, J=2.7 Hz, C═CH₂). ¹³C NMR (CDCl₃, 75 MHz) ppm 14.32 (C₃), 21.14(CH₃), 22.94 (C₂), 66.30 (CH₂O), 105.96 (═CH₂), 134.05 (C₁), 171.26(CO). CI-MS 199 (M+H, 0.27), 57 (100.0).

EXAMPLE 5 1,1-Bis-(acetoxymethyl-2-bromo-2-(bromomethyl)-cyclopropane(3) from 2,2-(bis-acetoxymethyl)methylenecyclopropane (3)

Bromine (3.2 g, 20 mmol) was added dropwise to a solution of compound 8(3.95 g, 20.0 mmol) from Example 4 in carbon tetrachloride (30 mL) withstirring at 0° C. The stirring was continued for 30 min. The reactionmixture was diluted with ethyl acetate (100 mL) and the organic phasewas washed with saturated aqueous solution of sodium thiosulfate andsodium hydrogen carbonate and then with water. After drying withmagnesium sulfate, the solvents were evaporated in vacuo and the residuewas chromatographed on a silica gel column (hexanes-ethyl acetate, 10:1and then 5:1) to give compound 3 as a white solid (4.15 g, 58%).

Mp. 56-58° C. ¹H NMR (CDCl₃, 400 MHz) δ 1.33 (d, 1H, J=7.2 Hz) and 1.46(d, 1H, J=7.2 Hz, H₂), 2.08 (s, 3H) and 2.10 (s, 3H, CH₃), 3.75 (d, 1H,J=11.2 Hz), 3.96 (d, 1H, J=11.2 Hz), 4.23 (m, 3H) and 4.48 (d, 1H, J=12Hz, CH₂Br+OCH₂). ¹³C NMR (CDCl₃, 100 MHz) ppm 21.12 (C₂), 27.22 (CH₃),32.08 (C₃), 41.47 (CH₂Br), 42.48 (C_(1′)), 62.32 and 68.12 (CH₂O),170.96 and 171.01 (CO). CI-MS 361, 359 and 357 (M+H, 21.3, 42.8 and22.0), 299 (100.0), 277 and 279 (M-Br, 68.2 and 68.0). EI-HRMScalculated for C₁₀H₁₄ ⁷⁹Br₂O₄—Br: 277.0075, found: 277.0074. Calculatedfor C₁₀H₁₄Br₂O₄: C, 33.55; H, 3.94; Br, 44.64. Found: C, 33.75; H, 4.10;Br, 44.80.

EXAMPLE 6 1,1-Bis-(acetoxymethyl-2-bromo-2-(bromomethyl)-cyclopropane(3) from a mixture of compounds 4 and 6

A mixture of compounds 4+6 (2.0 g, 10 mmol) from Example 2 was reducedwith lithium aluminum hydride in ether as described in Example 3. Theobtained mixture of diols 7 and 9 (866 mg, 76%) was used directly in thenext step.

¹H NMR (CDCl₃, 300 MHz) δ 1.20 (t, 2H, J=2.1 Hz, cyclopropane ofcompound 7), 1.76 (12H, CH₃ of compounds 7+9), 3.65 (AB, 4H, J=10.8 Hz,CH₂O of compound 7), 4.27 (s, 4H, CH₂O of compound 9), 5.38 (s, 1H) and5.47 (t, 1H, J=2.1 Hz, CH₂=of compound 7).

A mixture of compounds 7+9 (570 mg, 5 mmol) was acetylated using aceticanhydride in pyridine as described in Example 4 to give a 1:1 mixture ofacetates 8+10 (915 mg, 92%) which was used directly in the next step.

Compound 10: ¹H NMR (CDCl₃, 400 MHz) δ 1.82 (s, 6H, CH₃), 2.03 (s, 6H,CH₃ of acetyl), 4.65 (s, 4H, CH₂O). ¹³C NMR (CDCl₃, 100 MHz) ppm 21.06and 21.20 (CH₃), 62.71 (CH₂O), 123.12 and 141.19 (C═C), 171.35 (CO).

¹H NMR and ¹³C NMR of compound 8 were identical with those given inExample 4.

Pyridinium perbromide (1.60 g, 5 mmol) was added to a solution of amixture of compounds 8+10 (796 mg, 4 mmol) in dichloromethane (50 mL) at0° C. The reaction mixture was then allowed to stand at room temperaturefor 15 h. Ethyl acetate (100 mL) was then added and the organic phasewas washed with a saturated solution of sodium bicarbonate and sodiumthiosulfate followed by water. After drying with sodium sulfate, thesolvents were evaporated and the crude product was chromatographed on asilica gel column using hexanes-ethyl acetate (10:1).1,1-Bis(acetoxymethyl)-1,2-dibromo-2,2-dimethylethane (11) obtained as acolorless liquid was eluted first (529 mg, 37%) followed by compound 3(white solid, 659 mg, 46%).

Compound 11: ¹H NMR (CDCl₃, 400 MHz) δ 2.05 (s, 6H, CH₃ of acetyl), 2.15(s, 6H, CH₃), 4.69 (d, 4H, J=1.6 Hz, CH₂O). ¹³C NMR (CDCl₃, 400 MHz) ppm21.20 (CH₃ of acetyl), 33.06 (CH₃), 65.91 (CH₂O), 67.08 and 73.33(C—Br), 170.19 (CO).

Compound 3 was identical with the product described in Example 5.

EXAMPLE 7 (Z)-9-{[2,2-Bis-(acetoxymethyl)cyclopropylidene]methyl}-adenine (13a) and(E)-9-{[2,2-Bis-(acetoxymethyl)cyclopropylidene]-methyl}adenine (14a)

A mixture of adenine (12a, 3.17 g, 2.35 mmol), dibromide 3 (0.84 g, 2.35mmol) from Example 5 or 6 and flame-dried potassium carbonate (1.95 g,14.1 mmol) in N,N-dimethylformamide (20 mL) was stirred at 100° C. undernitrogen for 24 h. After cooling, the insoluble portion was filteredoff, it was washed with N,N-dimethylformamide and the filtrate wasevaporated in vacuo. The residue was chromatographed on a silica gelcolumn using dichloromethane-methanol (20:1) to give a mixture of E- andZ-isomers 13a and 14a (330 mg, 42%) as a white solid.

Mp 155-157° C. UV max (ethanol) 276 nm (E 7,400), 256 nm (s 10,800), 228nm (ε20,500). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.61 (s, 2H) and 1.79 (s, 2H,H_(3′)), 2.07 (s, 3H) and 2.10 (s, 31-1, CH₃), 4.10 (d, 2H, J=8 Hz),4.07 (d, 2H, J=8 Hz), 4.28 (d, 1H, J=11.2 Hz) and 4.43 (d, 1H, J=11.2Hz, H_(5′)), 6.05 (s, 2H) and 6.13 (s, 2H, NH₂), 7.56 (s, 1H) and 7.70(s, 1H, H_(1′)), 8.24 (s, 1H, H₂, Z-isomer), 8.38 (s, 2H, H₂+H_(g),E-isomer), 8.46 (s, 1H, H₈, Z-isomer). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm13.27 and 15.84 (C_(3′)), 21.03 and 21.15 (CH₃), 23.41 and 25.09(C_(4′)), 66.16 and 66.47 (C_(5′)), 113.03 (C_(1′)), 114.69 and 114.83(C_(2′) and C₅), 136.95 and 137.92 (C₈), 149.11 (C₄), 153.78 (C₂),155.83 (C₆), 170.73 and 171.10 (CO). EI-MS 331 (M, 10.1), 272 (99.5),230 (46.0), 200 (20.1), 136 (100.0), 135 (30.3), 95 (48.1). EI-HRMScalcd. for C₁₅H₁₇N₅O₄: 331.12805, found: 331.12806. Anal. Calcd. forC₁₅H₁₇N₅O₄: C, 54.38; IA, 5.17; N, 21.14. Found: C, 54.17; H, 5.23; N,21.29.

EXAMPLE 8(Z)-9-{(2,2-Bis-(hydroxymethyl)cyclopropylidene)methyl}-adenine (1a) and(E)-9-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]-methyl}adenine (2a)

A mixture of compounds 13a+14a (309 mg, 0.93 mmol) from Example 7 andpotassium carbonate (0.83 g, 6 mmol) in methanol-water (9:1, 30 mL) wasstirred at room temperature for 12 h whereupon TLC showed a completereaction. Acetic acid was carefully added and the mixture was evaporatedin vacuo. The residue was chromatographed in dichloromethane-methanol(10:1) to give Z- and E-isomers 1a and 2a.

Z-isomer 1a (87 mg, 38%): Mp. 239-242° C. UV max (ethanol) 276 nm(ε8,200), 262 nm (ε11,700), 227 nm (ε25,200). ¹H NMR (CD₃SOCD₃, 400 MHz)δ 1.34 (s, 2H, H_(3′)), 3.52, 3.68 and 3.53, 3.67 (2AB, ²J=11.0 Hz, 4H,H_(5′)), 5.07 (t, 2H, ³J=4.0 Hz OH), 7.37 (s, 1H, H_(1′)), 7.36 (s, 2H,NH₂), 8.17 (s, 1H, H₂), 8.82 (s, 1H, H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz)ppm 11.65 (C_(3′)), 31.41 (C_(4′)), 62.84 (C_(5′)), 111.12 (C_(1′)),118.51 (C_(2′)), 119.09 (C₅), 138.48 (C₈), 148.59 (C₄), 153.61 (C₂),156.69 (C₆). EI-MS 247 (M, 9.1), 230 (14.3), 200 (23.9), 136 (100.0),135 (56.0), 69 (24.2). EI-HRMS calculated for C₁₁H₁₃N₅O₂: 247.1069,found: 247.1069. Calculated for C₁₁H₁₃N₅O₂: C, 53.43; H, 5.30; N, 28.32.Found: C, 53.21; H, 5.33; N, 28.57.

E-isomer 2a (66 mg, 29%): Mp 250-252° C. UV max (ethanol) 276 nm(ε7,100), 260 nm (ε10,000), 227 nm (ε21,300). ¹H NMR (CD₃SOCD₃, 400 MHz)δ 1.56 (d, 2H, 2H, J=2 Hz, H_(3′)), 3.46, 3.52 and 3.48, 3.51 (partiallyoverlapped 2AB, 4H, ²J=11.0 and 11.2 Hz, H₅)—, 4.76 (t, 2H, ³J=4.8 Hz,OH), 7.48 (s, 1H, Hp), 7.37 (s, 2H, NH₂), 8.17 (s, 1H, H₂), 8.49 (s, 1H,H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 14.36 (C_(3′)), 29.68 (C_(4′)),63.06 (C_(5′)), 110.86 (H_(1′)), 119.36 (C_(2′)+C₅), 137.76 (C₈), 148.88(C₄), 153.72 (C₂), 156.71 (C₆). EI-MS 247 (M, 9.1), 230 (14.3), 200(23.9), 136 (100.0), 135 (56.0), 69 (24.2), EI-HRMS calculated forC₁₁H₁₃N₅O₂: 247.1069, found: 247.1070. Calculated for C₁₁H₁₃N₅O₂: C,53.43; H, 5.30; N, 28.32. Found: C, 53.23; H, 5.48; N, 28.44.

EXAMPLE 9(Z)-2-Amino-6-chloro-9-{[2,2-bis-(acetoxymethyl)cyclopropylidene]methyl}purine(13b) and(E)-2-Amino-6-chloro-9-{[2,2-bis-(acetoxymethyl)cyclopropylidene]methyl}purine(14b)

A mixture of 2-amino-6-chloropurine (12b, 393 mg, 2.30 mmol), dibromide3 (0.83 g, 2.32 mmol) from Example 5 or 6 and flame-dried potassiumcarbonate (1.90 g, 12.5 mmol) in N,N-dimethylformamide (15 mL) wasstirred at 100° C. under nitrogen for 24 h. After cooling, the insolubleportion was filtered off, and it was washed with N,N-dimethylformamide.The filtrate was evaporated in vacuo and the residue was chromatographedon a silica gel column using dichloromethane-methanol (98:2) to give amixture of Z- and E-isomers 13b and 14b (264 mg, 32%) as a white solid.

Mp. 215-216° C. UV max (ethanol) 311 nm (ε5,100), 230 nm (ε21,800), 204nm (ε13,700). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.64 (s, 2H, H_(3′),E-isomer), 1.89 (d, 2.8H, J=2.4 Hz, H_(3′), Z-isomer), 1.94 (s, 6H, CH₃,E-isomer), 2.04 (s, 8.4H, CH₃, Z-isomer), 4.06-4.15 (m, 2.8H of Z-isomerand 4H of E-isomer, H_(5′)) and 4.28 (d, 2.8H, J=11.2 Hz, Z-isomer, 7.02(s, 2H, NH₂, E-isomer), 7.06 (s, 2.8H, NH₂, Z-isomer), 7.30 (s, 2.8H,H_(1′), E-isomer), 7.40 (s, 1.4H, H_(1′), Z-isomer), 8.32 (s, 1H, H₈,E-isomer), 8.43 (s, 1.4H, H₈, Z-isomer). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm13.22 and 16.24 (C_(3′)), 21.13 and 21.33 (CH₃), 23.65 and 25.53(C_(4′)), 65.85 and 66.31 (C_(5′)), 112.36 and 112.65 (C_(1′)), 117.09and 117.15 (C_(2′)), 123.74 (C₅), 140.07 and 140.56 (C₈), 150.40 (C₄),153.21 and 153.11 (C₂), 160.76 (C₆), 170.72 and 170.99 (CO). EI-MS 365and 367 (M, 9.3 and 3.3), 306 (55.3), 308 (18.3), 170 (72.1), 172(24.2), 95 (56.6), 94 (33.1), 43 (100.0). EI-HRMS calculated for C₁₅H₁₆³⁵ClN₅O₄: 365.0891, found: 365.0888.

EXAMPLE 10(Z)-2-Amino-6-chloro-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}-purine(1b) and(E)-2-Amino-6-chloro-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(2b)

A mixture of Z- and E-isomers 13b+14b (260 mg, 0.71 mmol) from Example 9and potassium carbonate (78 mg, 0.57 mmol) in methanol-water (9:1, 10mL) was stirred for 30 minutes at room temperature. Acetic acid wascarefully added and the mixture was evaporated in vacuo. The residue waschromatographed on a silica gel column in dichloromethane-methanol(10:1) to give Z-isomer 1b (106 mg, 53%) and E-isomer 2b (75 mg, 37%).

Z-isomer 1b: Mp. 207-208° C. UV max (ethanol) 310 nm (ε7,900), 234 nm(ε27,800). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.34 (s, 2H, H_(3′)), 3.47, 3.67and 3.49, 3.66 (2AB, 4H, ²J=10.8 and 11.2 Hz, H_(5′)), 5.04 (poorlyresolved t, 2H, OH), 7.03 (s, 2H, NH₂), 7.18 (s, 1H, Hp), 8.81 (s, 1H,H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 11.72 (C_(3′)), 31.41 (C_(4′)),62.75 (C_(5′)), 110.61 (C_(1′)), 119.24 (C_(2′)), 123.75 (C₅), 140.56(C₈), 150.19 (C₄), 152.92 (C₂), 160.70 (C₆). ESI-MS (MeOH+NaCl) 282 and284 (M+H, 100.0 and 33.3), 304 and 306 (M+Na, 40.5 and 13.7), 585 and587 (2M+Na, 32.7 and 24.4). Calculated for C₁₁H₁₂ClN₅O₂: C, 46.90; 11,4.29; Cl, 12.59; N, 24.86. Found: C, 47.08; H, 4.35; Cl, 12.38; N,24.88.

E-isomer 2b: Mp. 230-234° C. (decomp.). UV max (ethanol) 310 nm(ε8,000), 234 nm (ε28,800). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.54 (s, 2H,H_(3′)), 3.41, 3.49 and 3.43, 3.47 (2AB, 4H, ²J=11.6 and 11.0 Hz,H_(5′))_(′), 5.42 (broad s, 2H, OH), 7.02 (s, 2H, NH₂), 7.30 (s, 1H,H_(1′)), 8.43 (s, 1H, H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 14.52(C_(3′)), 29.84 (C_(4′)), 62.96 (C_(5′)), 110.41 (C_(1′)), 120.53(C_(2′)), 123.71 (C₅), 140.11 (C₈), 150.26 (C₄), 153.17 (C₂), 160.68(C₆). ESI-MS (MeOH+NaCl) 282 and 284 (M+H, 100.0 and 32.1), 304 and 306(M+Na, 27.4 and 8.9), 585 and 587 (2M+Na, 6.7 and 11.3). Calculated forC₁₁H₁₂ClN₅O₂: C, 46.90; II, 4.29; Cl, 12.59; N, 24.86. Found: C, 47.10;H, 4.40; Cl, 12.40; N, 25.04.

EXAMPLE 11(Z)-9-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}-guanine (1c)

The solution of the Z-isomer 1b (100 mg, 0.36 mmol) from Example 10 informic acid (95-97%, 8 mL) was heated at 80° C. with stirring for 4 h.After cooling, the formic acid was evaporated in vacuo and the crudeproduct was dissolved in methanol (30 mL). A precipitated white solidwas stirred in methanolic ammonia (20%, 10 mL) at 0° C. for 4 h. Afterevaporation of volatile components, a suspension of the residue inmethanol (100 mL) was refluxed for 2 h. The mixture was kept overnightat 0° C. to give product 1c (83 mg, 89%).

Mp.>300° C. UV max (ethanol) 271 nm (ε11,500), 231 nm (ε26,400). ¹H NMR(CD₃SOCD₃, 400 MHz) δ 1.29 (s, 2H, H_(3′)), 3.48, 3.63 and 3.49, 3.62(2AB, 4H, ²J=10.8 and 11.2 Hz, 4H, ²J=10.8 and 11.2, H_(5′)), 4.99 (t,2H, J=5.6 Hz, OH), 6.52 (s, 2H, NH₂), 7.07 (s, 1H, H1′), 8.41 (s, 1H,H₈), 10.64 (s, 1H, NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 11.52 (C_(3′)),31.26 (C_(4′)), 62.75 (C_(5′)), 110.98 (C_(1′)), 116.92 (C_(2′)), 118.08(C₅), 135.13 (C₈), 150.29 (C₄), 154.56 (C₂), 157.38 (C₆). ESI-MS(MeOH+NaCl) 264 (M+H, 5.1), 286 (M+Na, 100.0), 549 (2M+Na, 41.1).Calculated for C₁₁H₁₃N₅O₃: C, 50.19; H, 4.98; N, 26.60. Found: C, 50.06;H, 5.09; N, 26.48.

EXAMPLE 12(E)-9-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}-guanine (2c)

The procedure described in Example 11 was used for the synthesis ofE-isomer 2c (59 mg, 84%) from compound 2b (75 mg, 0.27 mmol) fromExample 10.

Mp.>300° C. UV max (ethanol) 271 nm (ε12,500), 229 nm (ε31,800). ¹H NMR(CD₃SOCD₃, 400 MHz) δ 1.49 (s, 2H, H_(3′)), 3.41, 3.48 and 3.43, 3.47(2AB, 4H, ²J=11.6 and 11.0 Hz, H_(5′)), 4.76 (t, 2H, ³J=5.6 Hz, OH),6.58 (s, 2H, NH₂), 7.21 (s, 1H, H_(1′)), 8.03 (s, 1H, H₈), 10.77 (s, 1H,NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 14.26 (C_(3′)), 29.51 (C_(4′)),63.04 (C_(5′)), 110.78 (C_(1′)), 116.90 (C_(2′)), 118.93 (C₅), 134.27(C₈), 150.52 (C₄), 154.60 (C₂), 157.44 (C₆). ESI-MS (MeOH+NaCl) 264(M+H, 3.6), 286 (M+Na, 100.0), 549 (2M+Na, 33.0). Calculated forC₁₁H₁₃N₅O₃: C, 50.19; H, 4.98; N, 26.60. Found: C, 50.10; H, 5.04; N,26.89.

EXAMPLE 13(Z)-1-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}-cytosine (1e)and (E)-9-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]-methyl}cytosine(2e)

A mixture of N⁴-acetylcytosine (12d, 1.80 g, 5.0 mmol), dibromide 3 (766mg, 5.0 mmol) from Example 5 or 6 and flame-dried potassium carbonate(4.75 g, 30 mmol) in N,N-dimethylformamide (100 mL) was stirred at 100°C. under nitrogen for 12 h. The mixture was cooled to 50° C. andmethanol (5 mL) was added with stirring which was continued for 5 h.After cooling, the insoluble portion was filtered off and it was washedwith N,N-dimethylformamide. The filtrate was evaporated in vacuo and theresidue was chromatographed on a silica gel column indichloromethane-methanol (20:1 and then 4:1) to give a mixture ofproducts 1e+2e (680 mg, 61%) as a white solid in a ratio of 1:1.4.

¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.14 (s, 2H, H_(3′), Z-isomer), 1.40 (s,2.8H, H_(3′), E-isomer), 3.36-3.47 (m, 7.8H, H_(5′)), 4.70 (s, 2.8, OH,E-isomer), 4.95 (s, 2H, OH, Z-isomer), 5.78 (d, 1H, J=5.6 Hz, H₅,Z-isomer), 5.85 (d, 1.4H, J=6.0 Hz, H₅, E-isomer), 7.31 (s, 1H, H_(1′),Z-isomer), 7.38 (s, 1.4H, E-isomer), 7.45 (s, 2.8H, NH₂, E-isomer), 7.97(d, 1.4H, J=6.0 Hz, H₆, E-isomer), 8.24 (d, 1H, J=6.4 Hz, H₆, Z-isomer).¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 10.72 (C_(3′), Z-isomer), 13.64 (C_(3′),E-isomer), 27.46 (C_(4′), E-isomer), 31.08 (C_(4′), Z-isomer), 62.99(C_(5′), Z-isomer), 63.17 (C_(5′), E-isomer), 95.52 (C₅, Z-isomer),95.79 (C₅, E-isomer), 114.69 (C_(1′), Z-isomer), 115.32 (C_(1′),E-isomer), 115.70 (C_(2′), E-isomer), 116.56 (C_(2′), Z-isomer), 140.77(C₆, E-isomer), 141.12 (C₆, Z-isomer), 154.66 (C₄, Z-isomer), 154.87(C₄, E-isomer), 166.07 (C₂).

EXAMPLE 14 (Z)- and(E)-N⁴-Benzoyl-1-{[2,2-bis-(hydroxymethyl)-cyclopropylidene]methyl}cytosine(15) and (16)

A mixture of 1e+2e from Example 13 was dissolved in refluxing ethanol(100 mL). Benzoic anhydride (689 mg, 3.05 mmol) was added with stirringinto a hot solution and the refluxing was continued for 1 h. Five moreportions of benzoic anhydride (689 mg, 3.05 mmol each) were added everyhour. After cooling, the solvent was evaporated and the crude productwas chromatographed on a silica gel column in dichloromethane-methanol(20:1) to give Z-isomer 15 (380 mg, 38%) and E-isomer 16 (350 mg, 35%)as white solids.

Z-isomer 15: Mp 222-223° C. UV max (ethanol) 329 nm (ε14,200), 270 nm(ε19,200), 203 nm (e 23,900). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.28 (s, 2H,H_(3′)), 3.46, 3.66 and 3.48, 3.64 (2AB, 4H, J=10.8 and 11.4 Hz), 4.99(s, 2H, OH), 7.32 (d, J=8 Hz, 1H), 7.39-7.50 (m, 3H, H_(1′)+H_(meta)'sof benzoyl), 7.64 (m, 3H), 7.60 (t, 1H, J=8 Hz, H_(para) of benzoyl),7.98 (d, 2H, J=8 Hz, H_(ortho)'s of benzoyl), 8.70 (d, J=7.2 Hz, 1H,H₆), 11.30 (s, 1H, NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 10.96 (C_(3′)),31.53 (C_(4′)), 62.86 (C_(5′)), 97.35 (C₅), 116.41 (C_(1′)), 120.17(C_(2′)), 129.94 (C_(ortho) of benzoyl), 129.18 (C_(meta) of benzoyl),133.46 (C_(para) of benzoyl), 145.24 (C₆), 153.99 (C₄), 163.64 (C₂),168.25 (CO of benzoyl). ESI-MS (MeOH+NaCl) 328 (M+H, 100.0), 350 (M+Na,71.9), 677 (2M+Na, 52.1). Calculated for C₁₇H₁₇N₃O₄: C, 62.38; H, 5.23;N, 12.84. Found: C, 62.50; H, 5.41; N, 13.02.

E-isomer 16: Mp 221-223° C. UV max (ethanol) 329 nm (ε14,200), 269 nm(ε18,900), 203 nm (e 23,400). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.52 (s, 2H),3.44, 3.50 and 3.45, 3.48 (2AB, 41-1, ²J=11.2 and 11.4 Hz, H_(5′)),7.45-7.51 (m, 3H, H_(1′)+H_(meta)'s of benzoyl), 7.60 (d, 1H, J=7.2 Hz,of benzoyl), 7.99 (d, 2H, J=7.2 Hz, H_(ortho)'s of benzoyl), 8.46 (d,1H, J=7.2 Hz, H₆), 11.33 (s, 1H, NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm13.66 (C_(3′)), 28.16 (C_(4′)), 62.96 (C_(5′)), 97.66 (C₅), 115.46(C_(1′)), 120.77 (C_(2′)), 129.13 (C_(ortho) of benzoyl), 129.18(C_(meta) of benzoyl), 133.46 (C_(para) of benzoyl), 133.75 (C_(ipso) ofbenzoyl), 145.09 (C₆), 154.33 (C₄), 163.72 (C₂), 167.97 (CO of benzoyl).ESI-MS (MeOH+NaCl) 328 (M+H, 100.0), 350 (M+Na, 97.6), 677 (2M+Na,100.0). EI-HRMS calculated for C₁₇H₁₇N₃O₄: 327.1219, found: 327.1221.Calculated for C₁₇H₁₇N₃O₄: C, 62.38; H, 5.23; N, 12.84. Found: C, 62.14;H, 5.30; N, 12.63.

EXAMPLE 15(Z)-1-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}-cytosine (1e)

The Z-isomer 15 (297 mg, 0.91 mmol) from Example 14 was stirred inmethanolic ammonia (20%, 30 mL) at room temperature for 12 h. Thesolvent was evaporated and the crude product was chromatographed on asilica gel column in dichloromethane-methanol (4:1) to give the Z-isomer1e (174 mg, 86%) as a white solid.

Mp 250-253° C. UV max (ethanol) 297 nm (ε11,900), 230 nm (ε12,700), 206(ε13,700). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.14 (s, 2H, H_(3′)), 3.34 and3.57 (AB, 4H, ²J=11.0 Hz, H_(5′)), 5.02 (broad s, 2H, OH, 5.82 (d, 1H,J=7.2 Hz, H₅), 7.31 (s, 1H, H_(1′)), 7.43 and 7.55 (2s, 2H, NH₂), 8.27(d, 1H, J=7.2 Hz, H₆). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 10.75 (C_(3′)),31.07 (C_(4′)), 63.03 (C_(5′)), 95.69 (C₅), 114.95 (C_(1′)), 116.52(C_(2′)), 141.20 (C₆), 154.85 (C₄), 166.12 (C₂). ESI-MS (MeOH+NaCl) 224(M+H, 2.7), 246 (M+Na, 100.0), 469 (2M+Na, 81.0). EI-HRMS calculated forC₁₀H₁₃N₃O₃: 223.0957, found: 223.0953. Calculated for C₁₀H₁₃N₃O₃: C,53.80; H, 5.87; N, 18.82. Found: C, 53.71; H, 6.00; N, 18.75.

EXAMPLE 16(E)-9-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}-cytosine (2e)

The E-isomer 16 (263 mg, 0.80 mmol) from Example 14 was debenzoylatedusing the procedure described in Example 15 to give compound 2e (149 mg,83%).

Mp 249-251° C., UV max (ethanol) 298 nm (ε12,200), 229 nm (ε12,300), 206nm (ε11,900). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.39 (s, 2H, H_(3′)), 3.37,3.42 and 3.37, 3.41 (2AB, 4H, ²J=10.6 and 11.2 Hz, H_(5′)), 4.78 (poorlyresolved t, 2H, OH), 5.89 (d, 1H, J=8 Hz, H₅), 7.38 (s, 1H, H_(1′)),7.40 and 7.57 (2s, 2H, NH₂), 7.96 (d, 1H, J=7.4 Hz, H₆). ¹³C NMR(CD₃SOCD₃, 100 MHz) ppm 13.68 (C_(3′)), 27.47 (C_(4′)), 63.18 (CO, 95.92(C₅), 115.46 (CO, 115.66 (C_(2′)), 140.77 (C₆), 154.98 (C₄), 166.09(C₂). EI-MS (MeOH+NaCl) 224 (M+H, 2.7), 246 (M+Na, 100.0), 469 (2M+Na,81.0). Calculated for C₁₀H₁₃N₃O₃: C, 53.80; H, 5.87; N, 18.82. Found: C,54.01; H, 6.02; N, 18.72.

EXAMPLE 171-{[1-Bromo-2,2-bis-(acetoxymethyl)cyclopropyl]methyl}-thymine (18)

A mixture of 2,4-bis-(trimethylsilyloxy)-5-methylpyrimidine (17, 680 mg,2.50 mmol) and dibromoester 3 (0.90 g, 2.5 mmol) from Example 5 or 6 wasrefluxed in acetonitrile (20 mL) for 148 h. After cooling, ethanol (20mL) was added and solvents were evaporated. The residue was trituratedwith dichloromethane (50 mL), the insoluble portion was filtered offusing a bed of silica gel which was then washed withdichloromethane-methanol (30:1). The combined filtrate and washings wereevaporated. The crude product was chromatographed on a silica gel columnin dichloromethane-methanol starting from 100% dichloromethane andincreasing the amount of methanol to 40:1 to give compound 18 (750 mg,74.4%) as a white solid.

Mp 197-198° C. UV max (ethanol) 268 nm (ε10,400), 210 nm (ε8,900). ¹HNMR (CD₃SOCD₃, 400 MHz) δ 1.38 (d, 1H, J=7.2 Hz, 1H) and 1.72 (d, 1H,J=7.2 Hz, H_(3′)), 1.78 (s, 3H, 5-CH₃), 2.02 (s, 31-1) and 2.04 (s, 3H,CH₃ of acetyl), 4.10-4.17 (m, 3H) and 4.30-4.47 (m, 3H, H_(1′)+H_(5′)),7.54 (s, 1H, H₆), 11.35 (s, 1H, NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm12.76 (5-CH₃), 21.29 (C_(3′)), 24.99 (C_(4′)), 28.55 (CH₃ of acetyl),43.25 (C_(2′)), 52.47 (C_(1′)), 64.13 and 68.75 (C_(5′)), 109.18 (C₅),141.60 (C₆), 151.82 (C₂), 164.79 (C₄), 170.78 and 170.86 (CO of acetyl).EI-MS 405 and 403 (M, 4.4 and 4.4), 263 (6.2), 126 (10.9), 95 (9.4), 55(10.6), 43 (100.0). EI-HRMS calculated for C₁₅H₁₉ ²⁹BrN₂O₆: 402.0426,found: 402.0427. Calculated for C₁₅H₁₉BrN₂O₆: C, 44.68; H, 4.75; Br,19.82; N, 6.95. Found: C, 44.57; H, 4.89; Br, 19.83; N, 6.86.

EXAMPLE 18(Z)-1-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}-thymine (1f) and(E)-9-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]-methyl}thymine (2f)

A mixture of compound 18 (0.60 g, 1.49 mmol) from Example 17 andflame-dried potassium carbonate (616 mg, 4.47 mmol) inN,N-dimethylformamide (50 mL) was stirred at 100° C. under nitrogen for3 h. After cooling, methanol-water (9:1, 10 mL) was added with stirringwhich was continued at room temperature for 1 h. The insoluble portionwas filtered off and it was washed with N,N-dimethylformamide. Thefiltrate was evaporated in vacuo and the residue was chromatographed ona silica gel column which was first eluted with ethyl acetate and thendichloromethane-methanol (20:1) to give the Z-isomer 1f (70 mg, 38%) andE-isomer 2f (65 mg, 36%) as white solids.

Z-isomer 1f Mp 177-179° C. UV max (ethanol) 289 nm (ε11,700), 232 nm(ε12,800). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.17 (s, 2H, H_(3′)), 1.76 (s,3H, 5-CH₃), 3.40, 3.61 and 3.41, 3.60 (2AB, 4H, ²J=10.6 and 11.2 Hz,H_(5′)), 4.99 (t, J=6.0 Hz, 2H, OH), 7.17 (s, 1H, H_(1′)), 8.32 (s, 1H,H₆), 11.42 (s, 1H, NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 10.86 (C_(3′)),12.68 (5-CF₁₃), 31.18 (CO, 62.96 (C_(5′)), 111.39 (C_(1′)), 114.40(C_(2′)), 115.19 (C₅), 136.82 (C₆), 149.99 (C₂), 164.41 (C₄). EI-MS 238(M, 10.4), 221 (6.6), 127 (40.7), 126 (10.9), 113 (100.0), 83 (68.3), 55(26.6). EI-HRMS calculated for C₁₁H₁₄N₂O₄: 238.0954, found: 238.0953.Calculated for C₁₁H₁₄N₂O₄: C, 55.46; H, 5.92; N, 11.76. Found: C, 55.47;H, 5.96; N, 11.90.

E-isomer 2f: Mp 197-199° C. UV max (EtOH) 289 nm (ε11,000), 233 nm(ε12,100). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.47 (d, 2H, J=1.6 Hz), 1.82 (s,3H, 5-CH₃), 3.38, 3.45 and 3.40, 3.43 (2AB, 4H, ²J=11.2 and 11.4 Hz,H_(5′)), 4.66 (t, 2H, J=5.6 Hz, OH), 7.25 (s, 1H, H_(1′)), 7.82 (s, 1H,H₆), 11.46 (s, 1H, NH). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 12.82 (C_(3′)),13.94 (5-CH₃), 27.83 (C_(4′)), 63.09 (C_(5′)), 110.86 (C_(1′)), 113.77(C_(2′)), 116.33 (C₅), 136.12 (C₆), 150.20 (C₂), 164.38 (C₄). EI-MS 238(M, 12.8), 221 (27.5), 130 (17.7), 127 (100.0), 117 (19.4), 112 (74.9),83 (49.3). EI-HRMS calculated for C₁₁H₁₄N₂O₄: 238.0954, found: 238.0955.Calculated for C₁₁H₁₄N₂O₄: C, 55.46; H, 5.92; N, 11.76. Found: C, 55.62;H, 6.01; N, 11.88.

EXAMPLE 19(Z)-2-amino-6-methoxy-{[2,2-Bis-(hydroxymethyl)cyclopropylidene]methyl}purine(1g)

A solution of compound 1b (95 mg, 0.34 mmol) from Example 10 andpotassium carbonate (94 mg, 0.68 mmol) in methanol (15 mL) was refluxedfor 4 h. After cooling, the solvent was evaporated and the residue waschromatographed on a silica gel column using dichloromethane-methanol(10:1) to give the title compound 1g (86 mg, 91%).

Mp. 188-189° C. UV max (ethanol) 278 nm (ε10,400), 225 nm (ε26,900), 203nm (ε17,200). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.31 (s, 2H, H_(3′)), 3.49,3.66 and 3.51, 3.65 (2AB, 4H, ²J=11.0 and 10.4 Hz, H_(5′)), 3.95 (s, 3H,OCH₃), 5.03 (t, 1H, ³J=4.8 Hz), 6.53 (s, 2H, NH₂), 7.19 (s, 1H, H_(1′)),8.56 (s, 1H, H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 11.62 (C_(3′)), 31.30(C_(4′)), 53.96 (OCH₃), 62.83 (C_(5′)), 110.96 (CO, 114.12 (C_(2′)),117.70 (C₅), 137.27 (C₈), 153.09 (C₄), 160.77 (C₂), 161.37 (C₆). EI-MS277 (M, 23.1), 166 (100.0). Calculated for C₁₂H₁₅N₅O₃: C, 51.98; H,5.45; N, 25.26. Found: C, 52.08; H, 5.16; N, 25.18.

EXAMPLE 20 (E)-2-Amino-6-methoxy-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]methyl}purine (2g)

A mixture of compound 2b (140 mg, 0.50 mmol) from Example 10 andpotassium carbonate (276 mg, 2.0 mmol) in methanol (20 mL) was refluxedfor 2 h. The work-up followed the procedure for the Z-isomer 1gdescribed in Example 19 to give compound 2g (127 mg, 92%).

Mp. 179-180° C. UV max (ethanol) 279 nm (ε10,000), 224 nm (c 28,200),201 nm (ε21,200). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.51 (s, 2H, H_(3′)),3.43, 3.50 and 3.45, 3.49 (2AB, 4H, ²J=11.2 and 11.0 Hz, H_(5′)), 3.95(s, 3H, OCH₃), 4.71 (t, ³J=5.6 Hz, 2H, OH), 6.51 (s, 2H, H₂), 7.31 (s,1H, H_(1′)), 8.20 (s, 1H, H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 14.34(C_(3′)), 29.56 (C_(4′)), 53.9 (OCH₃), 63.10 (C_(5′)), 110.78 (C_(1′)),114.11 (C_(2′)), 118.60 (C₅), 136.54 (C₈), 153.35 (C₄), 160.78 (C₂),161.37 (C₆). EI-MS 277 (M, 3.0), 260 (M-OH, 8.7), 179 (100.0). EI-HRMScalcd. for C₁₂H₁₅N₅O₃: 277.1175, found: 277.1174. Calculated forC₁₂H₁₅N₅O₃: C, 51.98; H, 5.45; N, 25.26. Found: C, 52.21; H, 5.32; N,25.45.

EXAMPLE 21(Z)-2-Amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)-cyclopropylidene]methyl}purine(1h)

A solution of compound 1b (140 mg, 0.5 mmol) from Example 10 andcyclopropylamine (0.14 mL, 1.0 mmol) was stirred at room temperature for40 h. After cooling, the volatile components were evaporated and theresidue was chromatographed using CH₂Cl₂:methanol (10:1) to givecompound 1h (139 mg, 92%).

Mp. 195-196° C. UV max (ethanol) 286 nm (ε16,600), 224 nm (ε40,200). ¹HNMR (CD₃SOCD₃, 300 MHz) δ 0.54-0.57 (m, 2H) and 0.62-0.66 (m, 2H, CH₂ ofcyclopropyl), 1.28 (d, 2H, J=2.1 Hz, H_(3′)), 3.01 (s, 1H, CH ofcyclopropyl), 3.49, 3.63 and 3.51, 3.62 (2AB, 4H, ²J=11.0 and 10.8 Hz,H_(5′)), 5.00 (t, 2H, ³J=4.8 Hz, OH), 5.94 (s, 2H, 2—NH₂), 7.16 (s, 1H,H_(1′)), 7.36 (poorly resolved d, 1H, 6—NH), 8.40 (s, 1H, H₈). ¹³C NMR(CD₃SOCD₃, 75 MHz) ppm 7.19 (CH₂ of cyclopropyl), 11.61 (C_(3′)), 24.61(CH of cyclopropyl), 31.25 (C_(4′)), 62.89 (C_(5′)), 111.22 (C_(1′)),113.71 (C_(2′)), 116.77 (C₅), 135.00 (C₈), 156.59 (C₂), 161.07 (C₆).ESI-MS 303 (M+H), 325 (M Na), 605 (2M+Na), 627 (2M+Na). Calculated forC₁₄H₁₈N₅O₂: C, 55.62; H, 6.00; N, 27.80. Found: C, 55.79; H, 5.86; N,27.80.

EXAMPLE 22(E)-2-Amino-6-cyclopropylamino-9-{[2,2-bis-(hydroxymethyl)-cyclopropylidene]methyl}purine(2h)

The procedure described for the Z-isomer 1 h in Example 21 was followedwith the E-isomer 2b and cyclopropylamine (0.70 mL, 5 mmol, 50° C., 20h) to give compound 2h (130 mg, 86%).

Mp. 164-165° C. UV max (ethanol) 286 nm (ε16,300), 224 nm (c 37,900). ¹HNMR (CD₃SOCD₃, 300 MHz) δ 0.57 (s, 2H) and 0.61-0.66 (m, 2H, CH₂ ofcyclopropyl), 1.48 (s, 2H, H_(3′)), 3.00 (bs, 1H, CH of cyclopropyl),3.43, 3.50 and 3.45, 3.48 (2AB, 4H, ²J=11.4 and 11.0, H_(5′)), 4.71 (t,³J=5.9 Hz, 2H, OH), 5.91 (s, 2H, 2—NH₂), 7.30 (poorly resolved t, 1H,H_(1′)), 7.41 (bs, 1H, 6—NH), 8.04 (s, 1H, H₈). ¹³C NMR ppm 7.1 (CH₂ ofcyclopropyl), 14.3 (C_(3′)), 24.5 (CH of cyclopropyl), 29.4 (C_(4′)),63.2 (C_(5′)), 111.0 (C_(1′)), 113.67 (C_(1′)), 117.34 (C₅), 133.9 (C₈),150.7 (C₄), 156.6 (C₂), 161.2 (C₆). EI-MS 302 (M, 92.2), 285 (M-OH,35.0), 191 (100.0). EI-HRMS calcd. for C₁₄H₁₈N₅O₂: 302.1491, found:302.1491. Calculated for C₁₄H₁₈N₅O₂: C, 55.62; H, 6.00; N, 27.80. Found:C, 55.52; H, 5.96; N, 27.69.

EXAMPLE 23(Z)-2,6-Diamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}-purine(1o)

A mixture of compound 1b (140 mg, 0.5 mmol) from Example 10 and NH₃ inmethanol (saturated at 0° C., 60 mL) was heated in a stainless steelbomb at 100° C. for 20 h. After cooling, the volatile components wereevaporated and the residue was chromatographed on silica gel usingdichloromethane-methanol (4:1) to give the title compound 1o (111 mg,85%).

Mp. 249-250° C. UV max (ethanol) 280 nm (ε13,400), 220 nm (c 35,500). ¹HNMR (CD₃SOCD₃, 400 MHz) δ 1.27 (s, 2H, H_(3′)), 3.49, 3.62 and 3.50,3.61 (2AB, 4H, ²J=10.8 and 10.4 Hz, H_(5′)), 5.03 (t, 2H, ³J=4.8 Hz,OH), 5.85 (s, 2H, 2—NH₂), 6.74 (s, 2H, 6—NH₂), 7.13 (s, 1H, H_(1′)),8.39 (s, 1H, H₈). ¹³C NMR (CD₃SOCD₃, 100 MHz) ppm 11.61 (C_(3′)), 31.28(C_(4′)), 62.89 (C_(5′)), 111.24 (C_(1′)), 113.47 (C_(2′)), 116.65 (C₅),135.16 (C₈), 150.90 (C₄), 156.82 (C₂), 161.21 (C₆). EI-MS 262 (M, 19.6),150 (100.0). EI-HRMS calcd. for C₁₁H₁₄N₆O₂: 262.1178, found 262.1175.Calculated for C₁₁H₁₄N₆O₂: C, 50.38; H, 5.38; N, 32.04. Found: C, 50.49;H, 5.12; N, 32.24.

EXAMPLE 24(E)-2,6-Diamino-9-{[2,2-bis-(hydroxymethyl)cyclopropylidene]-methyl}purine(2o)

The procedure described for the Z-isomer 1o in Example 23 was performedon a 0.34 mmol scale of compound 2b to give the E-isomer 2o (72 mg,81%).

Mp. 219-220° C. UV max (ethanol) 280 nm (ε13,700), 220 nm (c 42,700). ¹HNMR 1.48 (d, 2H, J=2.4 Hz, H_(3′)), 3.43, 3.50 and 3.45, 3.48 (2AB, 4H,²J=11 Hz, H_(1′)), 4.68 (t, 2H, ³J=3.0 Hz, OH), 5.85 (s, 2H, 2—NH₂),6.77 (s, 2H, NH₂), 7.27 (t, 1H, J=2.4 Hz, H_(1′)), 8.04 (s, 1H, H₈). ¹³CNMR (CD₃SOCD₃, 100 MHz) ppm 14.27 (C_(3′)), 29.41 (C_(4′)), 63.19(C_(5′)), 110.99 (C_(1′)), 113.42 (C_(2′)), 117.42 (C₅), 134.22 (C₈),151.20 (C₄), 156.83 (C₂), 161.25 (C₆). EI-MS 262 (M, 26.9), 151 (100.0).EI-HRMS calcd. for C₁₁H₁₄N₆O₂: 262.1178, found 262.1172. Calculated forC₁₁H₁₄N₆O₂: C, 50.38; H, 5.38; N, 32.04. Found: C, 50.51; H, 5.13; N,32.30.

EXAMPLE 25(Z)-2-Amino-6-fluoro-9-{[2,2-bis-hydroxymethyl)cyclopropylidene]methyl}-purine(1p)

A mixture of the Z-isomer 1b (140 mg, 0.5 mmol) from Example 10, 1 Msolution of trimethylamine in N,N-dimethylformamide (0.21 mL, 0.21 mmol)and potassium fluoride (400 mg, 6.9 mmol, dried in vacuo at roomtemperature/0.05-0.07 torr for 12 h) in N,N-dimethylformamide (5 mL) wasvigorously stirred at room temperature for 24 h. The solids werefiltered off, they were washed with DMF and the filtrate was evaporatedin vacuo. The crude product was chromatographed on silica gel usingethyl acetate-methanol (50:1 to 30:1) to give the title compound 1p (113mg, 85%).

Mp. 185-188° C. UV max (ethanol) 289 nm (ε8,400), 268 nm (ε8,700), 299nm (ε37,000). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.34 (s, 2H, H_(3′)), 3.35,3.67 and 3.49, 3.66 (2AB, 4H, J_(AB)=10.2 Hz), 5.02 (poorly resolved t,2H, OH), 7.01 (s, 2H, NH₂), 7.20 (s, 1H, H_(y)), 8.77 (s, 1H). ¹³C NMR11.67 ppm (C_(3′)), 31.40 (C_(4′)), 62.78 (C_(5′)), 110.78 (C_(1′)),111.86 (C₅, d, J_(C,F)=31.3 Hz), 119.04 (C_(2′)), 140.22 (C₈), 156.37(C₄, J_(C,F)=12.0 Hz), 159.87 (C₆, d, ¹J_(C,F)=250.7 Hz), 160.70 (C₂, d,³J_(C,F)=17.9 Hz). ¹⁹F NMR (CD₃SOCD₃, 376 MHz) ppm −72.81(s). EI-MS 265(M, 3.8), 248 (M-OH, 5.3), 154 (M-purine base, 100.0). EI-HRMS calcd.for C₁₁H₁₂N₅O₂F: 265.0975, found: 265.0974. Calculated for C₁₁H₁₂N₅O₂F:C, 49.81; H, 4.56; N, 26.40. Found: C, 49.92; H, 4.70; N, 26.26.

EXAMPLE 26(E)-2-Amino-6-fluoro-9-{[2,2-bis-hydroxymethyl)cyclopropylidene]methyl}purine(2p)

The procedure described in Example 25 was performed with the E-isomer 2bfrom Example 10 (0.5 mmol scale) to give compound 2p (107 mg, 81%).

Mp. 214-216° C. UV max 289 nm (£ 8,600), 271 nm (ε8,800), 229 nm(ε38,400). ¹H NMR (CD₃SOCD₃, 400 MHz) δ 1.53 (d, 2H, J=1.6 Hz), 3.44,3.55 and 3.45, 3.49 (2AB, 4H, J_(AB)=11.2 Hz), 4.74 (t, 2H, OH, J=5.6Hz), 7.01 (s, 2H, NH₂), 7.33 (s, 1H, H_(1′)), 8.42 (s, 1H, H₈). ¹³C NMR(CD₃SOCD₃, 100 MHz) ppm 14.46 (C_(3′)), 29.78 (C_(4′)), 62.98 (C_(5′)),110.62 (C_(1′)), 111.87 (C₅, d, ²J_(C,F)=31.4 Hz), 120.26 (C_(2′)),139.79 (C₈), 156.64 (C₄, d, ³J_(C,F)=12.0 Hz), 159.88 (C₆, d,¹J_(C,F)=250.7 Hz), 160.70 (C₂, d, ³J_(C,F)=17.9 Hz). ¹⁹F NMR (CD₃SOCD₃,376 MHz) ppm −72.6 (s). EI-MS 265 (M, 2.8), 248 (M-OH, 4.0), 154(M-purine base, 100.0). EI-HRMS calcd. for C₁₁H₁₂N₅O₂F: 265.0975, found:265.0972. Calculated for C₁₁H₁₂N₅O₂F: C, 49.81; H, 4.56; N, 26.40.Found: C, 49.86; H, 4.68; N, 26.36.

EXAMPLE 27 In Vitro Antiviral Evaluation Methods

Cells and viruses. The routine growth and passage of KB cells wasperformed in monolayer cultures using minimal essential medium (MEM)with either Hanks salts [MEM(H)] or Earle salts [MEM(E)] supplementedwith 10% calf serum. The sodium bicarbonate concentration was varied tomeet the buffering capacity required. Cultures of diploid human foreskinfibroblasts (HFF) or MRC-5 cells were grown in medium consisting ofMEM(E) with 10% fetal bovine serum. Cells were passaged at 1:2 to 1:10dilutions according to conventional procedures by using 0.05% trypsinplus 0.02% EDTA in a HEPES buffered salt solution (HBS) (Shipman, C.,Jr., Proc. Soc. Exp. Biol. 130:305-310 (1969)) as described previously.Turk, S. R., et al., Antimicrob. Agents Chemother. 31:544-550 (1987).HFF and MRC-5 cells were passaged only at 1:2 dilutions. CEM cells weremaintained in suspension culture as detailed previously. Kucera, L. S.,et al., AIDS Res. Human Retroviruses 9:307-314 (1993).

Virological procedures. Stock HCMV was prepared by infecting HFF cellsat a multiplicity of infection (m.o.i.) of <0.01 plaque-forming units(p.f.u.) per cell. Cell growth medium was changed every four days untilcytopathology was evident in all cells (approximately 21 days).Supernatant fluids were retained as the virus stock. High titer HSV-1stocks were prepared by infecting KB cells at an m.o.i. of <0.1 asdetailed previously. Turk, S. R., et al., Antimicrob. Agents Chemother.31:544-550 (1987). Virus titers were determined using monolayer culturesof HFF cells for HCMV and monolayer cultures of BSC-1 cells for HSV-1 asdescribed earlier. Prichard, M. N. et al., J. Virol. Methods 28:101-106(1990). Briefly, HFF or BSC-1 cells were planted as described above in96-well cluster dishes and incubated overnight at 37° C. in a humidified3% CO₂-97% air atmosphere. The next day cultures were inoculated withHCMV or HSV-1 and serially diluted 1:3 across the remaining elevencolumns of the 96-well plate. Cultures were incubated at 37° C. for 2 hrto permit virus adsorption and then virus inoculum was replaced with 0.2mL of fresh medium. Cultures were incubated for seven days for HCMV, twoor three days for HSV-1, medium was removed, and the cell sheets werestained with 0.1% crystal violet in 20% methanol. Plaques wereenumerated under 20-fold magnification in wells having the dilutionwhich gave 5 to 20 plaques per well. Virus titers were calculatedaccording to the following formula: Titer (p.f.u./mL)=number ofplaques×5×3^(n); where n represents the nth dilution of the virus usedto infect the well in which plaques were enumerated.

Assays for Antiviral Activity. (a) HCMV. The effect of compounds on thereplication of HCMV has been measured using a plaque reduction assay.HFF cells in 24-well cluster dishes were infected with approximately 100p.f.u. of HCMV per cm² cell sheet using the procedures detailed above.Following virus adsorption, compounds dissolved in growth medium wereadded to duplicate wells in three to six selected concentrations.Following incubation at 37° C. for 7 to 10 days, cell sheets were fixed,stained with crystal violet and microscopic plaques enumerated asdescribed above. Drug effects were calculated as a percentage ofreduction in number of plaques in the presence of each drugconcentration compared to the number observed in the absence of drug.Ganciclovir (DHPG) was used as a positive control in all experiments.

The effect of compounds on the replication of HCMV may also be measuredusing a yield reduction assay. HFF cells were planted as described abovein 96-well cluster dishes, incubated overnight, medium removed and thecultures were inoculated with HCMV at a m.o.i. of 0.5 to 1 p.f.u. percell as reported elsewhere. After virus adsorption, inoculum wasreplaced with 0.2 mL of fresh medium containing test compounds. Thefirst row of 12 wells was left undisturbed and served as virus controls.Each well in the second row received an additional 0.1 mL of medium withtest compound at three times the desired final concentration. Thecontents of the 12 wells were mixed by repeated pipetting and thenserially diluted 1:3 along the remaining wells. In this manner, sixcompounds could be tested in duplicate on a single plate withconcentrations from 100 μM to 0.14 μM. Plates were incubated at 37° C.for seven days, subjected to one cycle of freezing and thawing; aliquotsfrom each of the eight wells of a given column were transferred to thefirst column of a fresh 96-well monolayer culture of HFF cells. Contentswere mixed and serially diluted 1:3 across the remaining eleven columnsof the secondary plate. Each column of the original primary plate wasdiluted across a separate plate in this manner. Cultures were incubated,plaques were enumerated, and titers calculated as described above.

Assays for Antiviral Activity. (b) HSV-1. An enzyme-linked immunosorbentassay (ELISA) was employed to detect HSV-1. 96-well cluster dishes wereplanted with BSC-1 cells at 10,000 cells per well, in a total volume of200 μL per well of MEM(E) plus 10% calf serum. After overnightincubation at 37° C., drug and HSV-1 was added at the rate of 100PFU/well. ELISA plates were blocked with 200 μL per well of 10% calfserum and 0.05% tween in HBS. After incubation for 30 minutes, theblocking agent was rinsed two times with HBS-T. A 1:400 dilution of APconjugated rabbit anti-HSV-1 antibody in HBS-F was added. Plates weresealed with adhesive sheet, and incubated on rocker for one hour at 37°C. Plates were developed in the dark with 1004 per well of substratesolution containing p-nitrophenyl phosphate. Plates were read at 492 nm.Drug effects were calculated as a percentage of the reduction in virusin the presence of each drug concentration compared to the titerobtained in the absence of drug. Acyclovir was used as a positivecontrol in all experiments.

Assays for Antiviral Activity. (c) HHV-6. In this case, enzyme-linkedimmunosorbent assay (ELISA) was performed in covalent amine plates(Costar, Cambridge, Mass.). The plates were activated by the addition ofa homobifunctional crosslinking agent, bis(sulfosuccinimidyl) suberate,which was dissolved at 1 mg/mL in 30 mL of phosphate buffered saline(PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 7.4)and 300 μL of the crosslinker was added to each well in the covalentplate. The crosslinker reacted with the amine function on the plate for30 min at room temperature. The byproduct, sodium N-hydroxysuccinimidesulfite, was removed by decanting and washing the plate twice with PBS.Samples consisting of 150 μl of mixed suspended HSB₂ cells from theoriginal drug-treated plate were solubilized in an equal volume of 10%Triton X-100 in coating buffer (15 mM Na₂CO₃, 3.5 mM NaHCO₃, pH 9.6).The plate was covered and then incubated for 1 h at 37° C. in a 5% CO₂atmosphere. These binding conditions facilitated covalent attachment ofthe antigen to the free end of the crosslinker.

After covalent binding, the antigen solution was decanted and the platewas washed six times in HEPES buffered saline (Shipman, C., Jr., Proc.Soc. Exp. Biol. 130:305-310 (1969)) with 0.05% Tween 20 (HBS-T), soakingfor three min for each wash. Unbound sites on the plate were blockedwith 300 μL per well of 2% lowfat dry milk in PBS (blocker) for 30 minat room temperature on a shaker. The blocker was decanted and 50 μL ofthe diluted primary monoclonal antibody, specific for HHV-6 (GS)glycoprotein gp116, was added. The antibody solution consisted ofantibody diluted 1:400 in equal volumes of blocker and 10% Triton X-100in coating buffer. The presence of both blocker and detergent in theantibody solutions was necessary to reduce background signal. The platewas then covered and incubated for 1 h at 37° C. The plate was washedagain, as described above, then blocker was added again, as before.Next, each well received 100 μL of a solution of the secondary antibody,horse radish peroxidase-labeled rabbit anti-mouse antibody, diluted to1:400 (as above). The plate was incubated for 1 h at 37° C. The platewas washed again as described above, and developed using 100 μL/well ofTMB-Turbo (Pierce, Rockford, Ill.) for 30 min at room temperature. Thereaction was stopped with 50 μL/well 2 M H₂SO₄. Absorbance in each wellwas determined at 450/570 nm.

Assays for Antiviral Activity. (d) HIV-1. Reverse transcriptase (RT) wasemployed as a marker for HIV-1. This assay measured the presence of HIVin supernatants of CEM cells infected with strain III_(B) of HIV-1 bythe amount of RT activity. Cells were grown, infected, and incubated inthe presence of seven concentrations (one-half log₁₀ dilutions)beginning at 1 or 100 μM of compounds to be assayed. Procedures and theRT assay were performed as detailed previously. Kucera, L. S., et al.,AIDS Res. Human Retroviruses 9:307-314 (1993); White, E. L., et al.,Antiviral Res. 16:257-266 (1991).

Cytotoxicity assays. Two different assays were used to explorecytotoxicity of selected compounds as we have detailed previously. (i)Cytotoxicity produced in stationary HFF cells was determined bymicroscopic inspection of cells used in plaque assays which were notaffected by the virus. Turk, S. R., et al., Antimicrob. AgentsChemother. 31:544-550 (1987). (ii) The effect of compounds during twopopulation doublings of KB cells was determined by crystal violetstaining and spectrophotometric quantitation of dye eluted from stainedcells. Prichard, M. N., et al., Antimicrob. Agents Chemother.35:1060-1065 (1991).

Data Analysis. Dose-response relationships were constructed by linearlyregressing the percent inhibition of parameters derived in the precedingsections against log drug concentrations. Fifty-percent inhibitory(IC₅₀) concentrations were calculated from the regression lines. Samplescontaining positive controls (acyclovir for HSV-1, ganciclovir for HCMV,and 2-acetylpyridine thiosemicarbazone for cytotoxicity) were used inall assays. Results from sets of assays were rejected if inhibition bythe positive control deviated from its mean response by >±1.5 standarddeviations.

Testing Results. Compounds of Formulas 1 and 2 exhibit a significantactivity against herpesviruses. It was found that compounds of thepresent invention strongly inhibit the replication of HMCV as measuredby plaque reduction assays using HFF as host cells by the methoddescribed above and they also inhibit the replication of HSV-1 asdetermined by enzyme-linked immunosorbent assay (ELISA).

TABLE 1 Compound IC₅₀ (μM) HCMV^(a) IC₅₀ (μM) HSV-1^(b) 1a, Example 83.6 50 1c, Example 11 0.46 >100 2c, Example 12 >100 39 1e, Example 1532 >100 1f, Example 18 >100 10 1g, Example 19 3.5 >100 1o, Example 2315 >100 Control 4.1^(c) 0.15^(d) ^(a)Plaque reduction. ^(b)ELISA.^(c)Ganciclovir. ^(d)Acyclovir.

The effects against HCMV are better than those of ganciclovir, currentdrug of choice for HCMV.

Compounds of the present invention were tested for cytotoxicity in aculture of HFF and KB cells according to the methods described above.These tests indicate a complete lack of cytotoxicity for testedcompounds that have antiviral activity.

TABLE 2 Compound IC50 (μM) HFF (visual) IC50 (μM) KB (growth) 1a,Example 8 >100 >100 1c, Example 11 >100 >100 2c, Example 12 >100 >1001e, Example 15 >100 >100 1f, Example 18 >100 >100 1g, Example19 >100 >100 1o, Example 23 >100 >100 Control   >100 ^(a)   >100 ^(b)^(a) Ganciclovir. ^(b) Acyclovir.

Those skilled in the art can now appreciate from the foregoingdescription that the broad teachings of the present invention can beimplemented in a variety of forms. Therefore, while this invention hasbeen described in connection with particular examples thereof, the truescope of the invention should not be so limited since othermodifications will become apparent to the skilled practitioner upon astudy of the drawings and specification.

All patents and other publications cited herein are expresslyincorporated by reference.

1. A compound having the formula:

wherein B is a 3-deazapurine, 7-deazapurine, 8-azapurine, or pyrimidine,and pharmaceutically acceptable salts and prodrugs thereof.
 2. Thecompound of claim 1, wherein B is selected from the group consisting of3-deazapurines, 7-deazapurines, and 8-azapurines.
 3. The compound ofclaim 1, wherein the pyrimidine is selected from the group consisting ofcytosine 5-halo substituted cytosines (wherein halo is fluoro, chloro,bromo, or iodo), thymines, uracils, 5-alkyl or alkenyl uracils, and6-azapyrimidines.
 4. The compound of claim 1, wherein the compound isselected from the group consisting of(Z)-1-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}cytosine(1e),(Z)-1-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}thymine(1f),(E)-1-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}cytosine(2e),and (E)-1-{[2,2-bis-(hydroxymethyl)cyclo-propylidene]methyl}thymine(2f).5. A composition comprising a compound of any one of claims 1-4 and apharmaceutically acceptable carrier.
 6. A method of treating of a mammalinfected with a virus selected from the group consisting of HHV, VZV,HCMV, EBV, HSV-1 and HSV-2, comprising a step of administering to saidmammal a compound according to any one of claims 1-4 and combinationsthereof.
 7. The method of claim 6, wherein said mammal is a human. 8.The method of claim 6, further comprising a step of administering anadditional compound.
 9. The method of claim 8, wherein said additionalcompound is selected from the group consisting of acyclovir,ganciclovir, zidovudine, AZT, ddl, ddc, 3TC, d4T, foscarnet, cidofovir,fomivirsen, and combinations thereof.